This project determines the structure of neuronal and glial cytoplasm, particu larly as it pertains to axoplasmic transport. A protein translocator, kinesin, is responsible for the anterograde organelle movements along microtubules which are the basis of anterograde fast axonal transport. A high molecular weight protein in squid axoplasm, which we have characterized as a cytoplasmic dynein, transports exclusively in the retrograde direction. Since organelles can bind both kinesin and dynein, the next question is how kinensin or dynein activation is programmed on the organelle surface. Metabolic uncouplers of various classes uniformly block organelle movements along microtubules in vitro, but do not block movements of latex beads induced by kinesin or dynein so it appears that an ionic gradient across the organelle membrane is responsible for programming an organelle to go in the anterograde or retrograde direction. The function of the transport system in vivo is also under investigation. Each organelle contacts several microtubules in the axon, so it is the continuous microtubule bundles which constitutes the transport pathways down the axon. Much of the pool of kinesin and dynein in vivo is in a soluble form and new immunocytochemical methods are being developed to determine their distributions in cytoplasm in relation to the transport pathways. An analysis of microtubule distributions in central nervous system cell bodies is underway. The numbers of organelles contacting, and therefore in transport, along these microtubules is being counted and it is apparent that there are different populations of microtubules, some supporting organelle transport in the cell body and some not. Turnover of the photoreceptive membranes, or rhabdom, was studied in Limulus photoreceptors maintained in vitro in order to investigate interactions of these membranes with the cytoskeleton. The distributions of actin and tubulin were examined by conventional and laser confocal fluorescence microscopy light-induced reduction of actin labeling suggests a role for actin in turnover of the microvilli, whereas microtubules may mediate the transport of shed microvilli.